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Recently, necroptosis has emerged as one of the important mechanisms of ischemia stroke. Necroptosis can be rapidly activated in endothelial cells to cause vascular damage and neuroinflammation. Panax notoginseng saponins (PNS), an ingredient extracted from the root of Panax notoginseng (Burk.) F.H. Chen, was commonly used for ischemic stroke, while its molecular mechanism and targets have not been fully clarified. Our study aimed to clarify the anti-necroptosis effect of PNS by regulating RIP1-RIP3-MLKL signaling pathway in brain microvascular endothelial cells (BMECs) subjected to transient oxygen-glucose deprivation (OGD/resupply [R]). In vitro, the necroptosis model of rat BMECs was established by testing the effect of OGD/R in the presence of the pan-caspase inhibitor z-VAD-FMK. After administration of PNS and Nec-1, cell viability, cell death modality, the expression of RIP1-RIP3-MLKL pathway and mitochondrial membrane potential (Δψm) level were investigated in BMECs upon OGD/R injury. The results showed that PNS significantly enhanced cell viability of BMECs determined by CCK-8 analysis, and protected BMECs from necroptosis by Flow cytometry and TEM. In addition, PNS inhibited the phosphorylation of RIP1, RIP3, MLKL and the downstream expression of PGAM5 and Drp1, while similar results were observed in Nec-1 intervention. We further investigated whether PNS prevented the Δψm depolarization. Our current findings showed that PNS effectively reduced the occurrence of necroptosis in BMECs exposed to OGD/R by inhibition of the RIP1-RIP3-MLK signaling pathway and mitigation of mitochondrial damage. This study provided a novel insight of PNS application in clinics.

Food fraud is currently a growing global concern with far-reaching consequences. Food authenticity attributes, including biological identity, geographical origin, agricultural production, and processing technology, are susceptible to food fraud. Metabolic markers and their corresponding authentication methods are considered as a promising choice for food authentication. However, few metabolic markers were available to develop robust analytical methods for food authentication in routine control. Untargeted metabolomics by liquid chromatography-mass spectrometry (LC-MS) is increasingly used to discover metabolic markers. This review summarizes the general workflow, recent applications, advantages, advances, limitations, and future needs of untargeted metabolomics by LC-MS for identifying metabolic markers in food authentication. In conclusion, untargeted metabolomics by LC-MS shows great efficiency to discover the metabolic markers for the authenticity assessment of biological identity, geographical origin, agricultural production, processing technology, freshness, cause of animals’ death, and so on, through three main steps, namely, data acquisition, biomarker discovery, and biomarker validation. The application prospects of the selected markers by untargeted metabolomics require to be valued, and the selected markers need to be eventually applicable at targeted analysis assessing the authenticity of unknown food samples.

The detection of rosiglitazone (RSG) in food is of great importance since the excessive intake of RSG could cause adverse effects on the human body. Although liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry are the preliminary methods for the detection of hazardous materials in food, they are not suitable for point-of-care or on-site detection. Herein, a time-based readout (TBR) device with an application software (APP) controlled by a smart phone was developed for the sensitive and selective immunoassay of RSG. The homemade TBR device was based on a two-electrode system, where the immune molecule-modified glassy carbon electrode was used as the bioanode, and Prussian blue-modified FTO was used as the cathode. By using Au-modified octahedral Cu

The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g���1 and a linear range from 0.5 to 80 ng g���1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g���1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry.

Mycotoxins are toxic contaminants in foods and feeds that are naturally occurring and largely unavoidable. Determining their contents in these products is essential to protect humans from harm. Immunoassays of mycotoxins have been well-established because they are fast, sensitive, simple, and cost-effective. However, a major limitation of immunoassays is the requirement of toxic mycotoxins as competing antigens, standards, or competing tracers. Mimotopes are peptides or proteins that can specifically bind to antibodies and compete with analytes for binding sites by mimicking antigenic epitopes. They can be employed as substitutes for competing antigens, standards, or competing tracers to avoid use of mycotoxins. This review summarizes the production and functionalization of the two main kinds of mimotopes, mimic peptides and anti-idiotypic antibodies (Ab2), and their applications in rapid analysis of mycotoxins.

The abnormal expression of microRNA (miRNA) can affect the RNA transcription and protein translation, leading to tumor progression and metastasis. Currently, the accurate detection of aberrant expression of miRNA, particularly using a portable detection system, remains a great challenge. Herein, a novel dual-mode biosensor with high sensitivity and robustness for miR-21 detection was developed based on the cis-cleavage and trans-cleavage activities of Cas12a. miRNA can be combined with hairpin DNA-horseradish peroxidase anchored on a CdS/g-C3N4/B-TiO2 photoelectrode, thus the nonenzymatic amplification was triggered to form numerous HRP-modified double-stranded DNA (HRP-dsDNA). Then, HRP-dsDNA can be specifically recognized and efficiently cis-cleaved by Cas12a nucleases to detach HRP from the substrate, while the remaining HRP on HRP-dsDNA can catalyze 4-chloro-1-naphthol (4-CN) to form biocatalytic precipitation (BCP) on the surface of the photoelectrode, and thus the photocurrent can be changed. Meanwhile, the trans-cleavage ability of Cas12a was activated, and nonspecifically degrade the FQ-reporter and a significant fluorescence signal can be generated. Such two different kinds of signals with independent transmission paths can mutually support to improve the performance of the detection platform. Besides, a portable device was constructed for the point-of-care (POC) detection of miR-21. Moreover, the dual-mode detection platform can be easily expanded for the specific detection of other types of biomarkers by changing the sequence of hairpin DNA, thereby promoting the establishment of POC detection for early cancer diagnosis.

In this work, an untargeted and pseudotargeted metabolomics combination approach was used for authentication of three shrimp species (Litopenaeus vanmamei, Penaeus japonicus, and Penaeus monodon). The monophasic extraction-based untargeted metabolomics approach enabled comprehensive-coverage and high-throughput analysis of shrimp tissue and revealed 26 potential markers. The pseudotargeted metabolomics approach confirmed 21 markers (including 9 key markers), which realized at least putative identification. The 21 confirmed markers, as well as 9 key markers, were used to develop PLS-DA models, correctly classifying 60/60 testing samples. Furthermore, DD-SIMCA and PLS-DA models were integrated based on the 9 key markers, with 59/60 and 20/20 samples of the species that were involved and uninvolved in model training correctly classified. The results demonstrated the potential of this untargeted and pseudotargeted metabolomics combination approach for shrimp species authentication.

Aminopyrine is a nonsteroidal anti-inflammatory drug only for medical purposes, however, it has been illegally added in traditional Chinese herbal teas for fraud activity recently. In this study, a specific antibody against aminopyrine with IC

Herbal tea is a highly popular and widely consumed beverage. However, a pain-relieving and fever-reducing drug, acetophenetidin, was recently found to illegally occur in herbal tea for a fraud purpose. Due to the potential health risk and urgent requirement for on-site screening method, a one-step and high specificity strip for identifying acetophenetidin was developed for the first time. Assisted by computational chemistry, four haptens were designed to prepare immunogens and coating antigens for antibody generation, and a specific antibody with ultra-sensitivity and high specificity was generated, showing half maximal inhibitory (IC50) of 16.46 ng/mL for acetophenetidin, less than 3.5% of cross-reactivity to analogs by ELISA. A gold nanoparticles immunochromatographic strip was developed for detection of acetophenetidin in herbal tea, demonstrating a cut-off value of 160 ng/mL and a limit of detection of 1.63 ng/mL. The recovery rates were ranged from 102.2% to 106.1% with coefficient of variation between 2.21% and 7.20%. The analysis of real samples (n = 20) by the strip was well correlated with that of the confirmatory method, liquid chromatography−tandem mass spectrometry. The proposed strip has the potential to be used for rapid screening of acetophenetidin in herbal tea.

Bisacodyl, sodium picosulfate and their metabolite bis-(p-hydroxyphenyl)-pyridyl-2-methane (BHPM) are clinically used to treat constipation. In this study, with the rational hapten design, a broad-specific and highly sensitive monoclonal antibody (mAb) was obtained with half of inhibitory concentrations of 0.16, 0.18 and 0.65 ng/mL for bisacodyl, sodium picosulfate and BHPM, respectively. Based on this mAb, a rapid and sensitive lateral flow immunochromatographic assay toward bisacodyl, sodium picosulfate and BHPM in slimming foods was developed. This method can qualitatively and quantitatively screen three stimulant laxatives with cut-off values of 3-6 ng/mL by naked eye and quantitative detection limits of 0.14-0.41 ng/mL by reader. The acceptable recoveries of spiked samples (78.00 %-120.12 %) and consistent results with liquid chromatography with tandem mass spectrometry (LC-MS/MS) in real sample detection confirmed the accuracy of the method. The established method provides a technique for multiplex, sensitive, fast, and on-site detection of three stimulant laxatives in slimming food.

Enantioselective antibodies have great potential to separate and detect chiral compounds. However, cross-reactivity of enantioselective antibodies to the distomer may limit the application. An in-depth understanding of interactions between antibodies and chiral drugs could be helpful to investigate antibody recognition to the distomer. In this study, a monoclonal antibody against chiral quinolone

Abstarct In the last few years, sildenafil and its analogues have emerged as an illegal ingredient in health food and products, causing illness and death. To address this problem, herein, a gold nanoparticle-based immunochromatographic assay for simultaneous detection sildenafil and its analogues was developed by using just one monoclonal antibody. This method can realize qualitative and quantitative analysis of sildenafil and its analogues with a detection range (IC20-IC80) of 0.12–28.05 ng mL−1, the IC50 was 0.31–8.45 ng mL−1 and the cut-off value was 5.0–80.0 ng mL−1. Meanwhile, this method could also accomplish semi-quantitative analysis of vardenafil with a cut-off value of 0.625 ng mL−1. The detection can be finished in 6 min and showed no cross-reactivity with avanafil and tadalafil. The recoveries of spiked samples ranged from 70.5 to 118.9% with a coefficient variation less than 15%, which confirmed the assay was sensitive, accurate and adapted to the rapid determination of sildenafil and its analogues.

Penicillium digitatum is the primary pathogen causing the green mold of citrus. The need for the development of higher effective and lower toxic natural antifungal agents is urgent, owing to the lack of antifungal agents that can successfully combat P. digitatum. Herein, the effects and mechanisms of 2-methoxy-1,4-naphthoquinone (MNQ) as a potential inhibitor of P. digitatumwere studied. First, MNQ showed a significant anti-P. digitatum effect with a minimum inhibitory concentration value of 5.0 μg/mL. Then, transcriptome, proteome, and metabolome profiling were performed on the MNQ-treated P. digitatum. A total of 910 genes, 297 proteins, and 174 metabolites changed significantly. The omics analysis indicated that it could be seen that MNQ mainly inhibits the growth of P. digitatum by affecting the synthesis of branched-chain amino acids and cell walls. These findings will be a great contribution to the further understanding of the possible molecular action of MNQ.

The coexistent pollution of multiple mycotoxins displays a synergistic toxicity effect that significantly threatens human health. Therefore, it is essential to establish a rapid detection method for multi-mycotoxins in food. In this study, red, green, and blue latex microspheres (LMs) were applied as the aflatoxin B

Herein, a novel immunochromatographic assay (ICA) based on metal-organic framework-decorated polydopamine (MOF@PDA) was firstly developed for the determination of hydrochlorothiazide (HCTZ) adulteration in functional foods. The coupling rate of MOF@PDA carrier to HCTZ antibody was as high as 91.7 %. The detection limits of the developed MOF@PDA-ICA in functional tablets and capsules were 5.93 and 4.72 μg/kg, the linear ranges were 11.2-91.91 μg/kg and 9.11-86.78 μg/kg, respectively. The sensitivity was 27-fold higher than that of the reported ICA. The recovery was 82.5-116.6 %, and coefficient of variation was 6.9-14.2 %. The results can be achieved and analyzed in 8 min with the smartphone-based detection device. The parallel tests of 23 commercial functional tablets and capsules showed that the results of the MOF@PDA-ICA were consistent with that of the LC-MS/MS (R

In this study, bifunctional magnetic ZnCdSe/ZnS quantum dots nanocomposite (MQNs) were synthesized, and firstly used to develop a lateral flow immunoassay (LFIA) for streptomycin (STR) and dihydrostreptomycin (DHSTR) detection in milk, muscle, liver, kidney, and honey simultaneously. The fluorescence signal of MQNs was 9-fold stronger than that of the original quantum dots. The detection limits of the established MQNs-LFIA for STR and DHSTR in five samples were 0.08-1.78 μg/kg, the quantitation limits were 0.26-5.87 μg/kg, the recoveries were between 85.0% and 120.0%, and the coefficient of variations were between 0.8% and 19.3%, respectively. The sensitivity was up to 42-fold more sensitive than the reported LFIAs. The single blind test results of 25 samples were consistent with that of the confirmation method (R

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Recently, increasing the number and severity of the natural hazards requires the resilience assessment and enhancement of the power system, especially the microgrid system. The emergency distribution generations have great potential to enhance the resilience of microgrid against blackouts under the emergency environment. This paper investigates the resilience assessment of the microgrid system under natural hazard, where emergency distribution generations are firstly pre-positioned on system nodes and then reconfigured after system damage occurs. A new resilience metric index and an efficient approximation computation method are provided for the resilience assessment of the focused problem. A new pre-position strategy and a new reconfiguration strategy on emergency distribution generations are proposed for the microgrid system emergency restoration under natural hazard. Also, a framework of resilience assessment is provided for problem-solving. The results of extensive experiments on the modified IEEE 30-Bus system and modified IEEE 118-Bus system confirm the effectiveness of the resilience assessment methodology and the superiority of proposed restoration strategies.

Three kinds of immunochromatographic assays (ICAs) are proposed for the highly sensitive and rapid determination of tylosin (TYL) and tilmicosin (TIM) in eggs based on colloidal gold (CG), latex microsphere (LM), and time-resolved fluorescent microsphere (TRFM). Three types of ICAs could tolerate the egg matrix via simple sample pretreatment and demonstrated high sensitivity for TYL and TIM with cut-off values of 6/6/3 μg/kg and 14/14/6 μg/kg, respectively. Furthermore, in a single-blind parallel study 20 egg samples were analyzed by the three developed ICAs and confirmed by LC-MS/MS. The results showed good consistency, and there were no false positive and false negative results in our three ICAs. Consequently, the proposed three ICAs offered rapid, highly sensitive, reliable, and selectable testing platforms for screening veterinary medicine or other small molecule contaminants.

Ochratoxin A (OTA), which has strong hepatotoxicity and nephrotoxicity, can accumulate in the human body through the food chain; thus, the selective and effective detection of OTA is urgently required for food security. Nanozymes with hyperfine size and shape control have attracted attention because of their controllable structure and intrinsic activity. Herein, CuFe-bimetal coordinated N-doped carbon (Cu@Fe-NC) with morphology-driven peroxidase-mimicking activity was synthesized using Cu

Semaphorin 4D (Sema4D) is highly expressed in various cancers and leukemia. It is involved in the development of acute leukemia. A high level of soluble Sema4D is also present in the plasma of acute leukemia patients. However, it remains unknown whether Sema4D is associated with the clinical characteristics of acute leukemia. In this study, Sema4D expression was examined in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) of patients with acute leukemia, and it was highly expressed in the PBMCs of B-acute lymphoblastic leukemia (ALL), T-ALL, and acute myeloid leukemia (AML) patients and in the BMMCs of B-ALL and AML patients but not in the BMMCs of T-ALL patients. Sema4D expression was higher in the PBMCs of T-ALL patients than in the PBMCs of B-ALL or AML patients. In addition, Sema4D expression in BMMCs was reduced in B-ALL patients during the chemotherapy process. It was lower in remission patients than in newly diagnosed and patients without remission. In acute leukemia, soluble Sema4D level in serum is positively correlated with Sema4D expression in PBMCs, leukocyte number, and peripheral blast number. Those results suggest that the levels of Sema4D and its soluble form are associated with acute leukemia development and may be regarded as a potential biomarker in pediatric acute leukemia.

Streptomycin (STR) and dihydrostreptomycin (DHSTR) are clinically widely used in the prevention and treatment of animal diseases. Unreasonable use or abuse can easily cause adverse effects on human health. Thus, it is very necessary to establish a rapid detection method for STR and DHSTR in animal-derived foods. In this study, a time-resolved fluorescent microspheres lateral flow immunoassay (TRFM-LFIA) integrated with a portable fluorescence reader was developed for STR and DHSTR detection. The cut-off values of the TRFM-LFIA for STR and DHSTR in milk/honey/muscle/liver/kidney were both 30/15/80/25/60 μg kg

Rapid, accurate, and safe screening of foodborne pathogenic bacteria is essential to effectively control and prevent outbreaks of foodborne illness. Fluorescent sensors constructed from carbon dots (CDs) and nanomaterial-based quenchers have provided an innovative method for screening of pathogenic bacteria. Herein, an ultrasensitive magnetic fluorescence aptasensor was designed for separation and detection of Staphylococcus aureus (S. aureus). Multicolor fluorescent CDs with a long fluorescent lifetime (6.73 ns) and high fluorescence stability were synthesized using a facile hydrothermal approach and modified cDNA as a highly sensitive fluorescent probe. CD fluorescence was quenched by Fe3O4 + aptamer via fluorescence resonance energy transfer (FRET). Under optimal conditions, the FRET-based aptasensor can detect S. aureus accompanied by a wide linear range of 50-107 CFU·mL-1 and a detection limit of 8 CFU·mL-1. Compared with other standard methods, this method was faster and more convenient, and the entire test was finished within 30 min. The capability of the aptasensor was simultaneously investigated on food samples. Additionally, the developed CDs exhibited excellent biocompatibility and were thus applied as fluorescent probes for bioimaging both in vitro and in vivo. This new platform provided an excellent application of the CDs for detecting and bioimaging pathogenic bacteria.

The development of rapid and in situ detection method for microcystin-LR (MC-LR) with portable, reliable, and easy to use devices are highly demanded, but it is still deficient. In this study, a portable and wireless point-of-need detection system for microcystin-LR (MC-LR) quantitation was developed and validated. The whole system consisted of three main parts: screen printed carbon electrodes (SPCEs), smartphone-controlled electrochemical analyzer (SCEA), and Android's smartphone. The SCEA was a homemade ARM STM32 microcontroller-based electrochemical analyzer, which have similar performance compared with a commercially available electrochemical workstation. A smartphone installed with a user-friendly application was employed to control the analyzer, receive and analyze data, and display detection results in real-time. This system can accurately quantitate MC-LR in the range of 0.001–100 μg/L with a detection limit of 0.00011 μg/L, and the results were consistent with LC–MS/MS. In addition, a preliminary MC-LR contamination map was generated using this smartphone-based platform by measuring water samples at 6 reservoirs located in Pearl River Delta (Guangdong Province, China). The cost-effective, reliable and easy-to-use smartphone-based detection system reported here can facilitate active toxicant screening to guarantee the safety of the drinking water, particularly in resource-limited area.

To monitor illegal addition of tadalafil and its analogues in health food, a new polyclonal antibody was prepared by immunizing New Zealand white rabbits with a common skeleton structure hapten combined with an unsaturated linear alkyl arm hapten strategy, which could simultaneously recognize tadalafil, amino tadalafil and N-Desmethyl tadalafil. A microwell lateral flow immune- chromatographic assay (mwLFIA) method combined with simple sample pretreatment was developed. The mwLFIA showed that the LOD for three tadalafil drugs was between 0.05 ng mL−1 and 0.30 ng mL−1, the cut-off values were between 50 ng mL−1 and 100 ng mL−1. Cross-reactivity (CR) results showed no interference from substances with similar structures, indicating a good specificity against tadalafil, amino tadalafil and N-Desmethyl tadalafil. The average recoveries of three drugs in spiked health wine and oral liquid ranged from 78.7% to 117.8% with an RSD of less than 15%, showing a good aggreement with the results of HPLC-MS/MS method. The developed mwLFIA strip was suitable for the rapid detection of tadalafil and its analogues in health wine and oral liquid samples.

In our study, a 3D printed smartphone-based detection device integrated with solid phase latex microsphere immunochromatography platform (SIAP) is developed for sensitive and quantitative detection of zearalenone (ZEN) in cereals and feed. A user-friendly Android application (App) is self-written to analyze, report and share the detection results. The cut-off values of latex microsphere immunochromatography for ZEN in cereals and feed were 2.5 and 3.0 μg/kg, respectively. The SIAP detection system can quantitatively detect ZEN in cereals and feed with the detection limits of 0.08 and 0.18 μg/kg, respectively. The recovery rates of corn, and feed samples were between 92.0%–105.0%, and 86.0%–107.5%, respectively, with the coefficient of variation ranging from 2.7% to 8.9%, and 7.8 to 10.9%, respectively. The detection results of real samples showed that there were differences in the content of ZEN in different feed ingredients and compound feed in China in October 2018, among which corn and wheat were still the feed ingredients with the most serious ZEN contamination, and it also demonstrated that the established SIAP detection system was reliable, portable, and user-friendly. This smartphone-based detection system will be popularized for on-site monitoring of various hazard factors such as veterinary drugs, antibiotics, illegal additives, etc.

Opium poppy abused in food has aroused public concerns due to its serious side effects. Effective monitoring is essential to fight the abuse crisis. Herein, we synthesized an easily prepared, affordable, accessible highly aggregated gold nanoparticles (AGNPs) performing in lateral flow immunoassay (LFIA) for detection opium poppy in herbal teas. Simultaneously, a LFIA based ontime-resolved fluorescent microspheres (TRFMs) was developed as contrastive method. In this study, morphine (MOR), codeine (COD) and thebaine (THE) were as the specific recognition markers of opium poppy. Results demonstrated the quantitative limits of detection were 0.0049/0.0053/0.084, 0.034/0.037/0.37 ng mL

In this study, an enhancing immunochromatography assay (ICA) based on colloidal gold-decorated polydopamine (CG@PDA) was firstly developed for gentamicin in milk, muscle, liver and kidney simultaneously. CG@PDA was synthesized by one-pot method with better signal intensity, colloidal stability, and antibody coupling efficiency than traditional colloidal gold. The detection limit of the developed ICA was about 1.50 μg/kg for the four animal-derived food, which was up to 92-fold more sensitive than the reported ICA based on colloidal gold. The recovery rates were ranged from 86.0% to 114.0% with coefficient of variation between 1.6% and 13.1%. Parallel analysis of 40 samples by commercial ELISA kits was confirmed the reliability. Our research indicated that polydopamine decorated can chemically modify the surface of colloidal gold and can thus remarkably improve the detection performances of ICA.